Devices, systems, and methods for detecting nucleic acids using sedimentation

ABSTRACT

Embodiments of the present invention are directed toward devices, systems, and method for conducting nucleic acid purification and quantification using sedimentation. In one example, a method includes generating complexes which bind to a plurality of beads in a fluid sample, individual ones of the complexes comprising a nucleic acid molecule such as DNA or RNA and a labeling agent. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a density lower than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a divisional of U.S. application Ser. No.13/423,008, filed Mar. 16, 2012, which in turn is a continuation-in-partof U.S. application Ser. No. 12/891,977, filed Sep. 28, 2010, entitled“Devices, systems, and methods for conducting sandwich assays usingsedimentation,” which application claims the benefit of the earlierfiling dates of U.S. Provisional Applications 61/362,398 filed Jul. 8,2010 and 61/362,407 filed Jul. 8, 2010. All afore-mentioned applicationsare hereby incorporated by reference, in their entirety, for anypurpose.

STATEMENT REGARDING RESEARCH & DEVELOPMENT

Described examples were made with Government support under Contract No.DE-NA0003525 awarded by the United States Department of Energy/NationalNuclear Security Administration. The Government has certain rights inthe invention, including a paid-up license and the right, in limitedcircumstances, to require the owner of any patent issuing in thisinvention to license others on reasonable terms.

TECHNICAL FIELD

Embodiments of the invention relate generally to assay systems andexamples include methods, systems, and apparatus employing sedimentationforces for conducting assays, including the detection and/orquantification of cell-free DNA or other nucleic acids.

BACKGROUND

Elevated presence of cell-free DNA (CFD) in blood has been linked tocancers, sepsis, radiation poisoning, brain damage, and stroke. The useof CFD as a biomarker for diagnosing such conditions has been inhibitedby the complexity and expense of current methods for determining theconcentration. Methods for determining CFD concentration in biologicalsamples such as serum include labeling with DNA responsive fluorescentdyes such as PicoGreen® from Life Technologies. Although capable ofmeasuring DNA directly in serum, such methods suffer from highbackground noise signal and reduced sensitivity due to the presence ofserum which exhibits substantial and variable autofluorescence.Moreover, spectroscopic determination of DNA concentration may typicallybe performed using UV absorbance, however, serum components also absorbat the relevant wavelengths, which complicates the analysis. Alternativehigh sensitivity methods for measuring CFD typically involve labor- andtime-intensive steps including DNA extraction and purification followedby gel electrophoresis and/or quantitative real-time polymerase chainreaction (qPCR).

Microfluidic systems, including “lab on a chip” or “lab on a disk”systems continue to be in development. See, Lee, B. S., et. al., “Afully automated immunoassay from whole blood on a disc,” Lab Chip 9,1548-1555 (2009) and Madou, M. et. al., “Lab on a CD,” Annu. Rev.Biomed. Engr. 8, 601-628 (2006), which articles are hereby incorporatedby reference in their entirety for any purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart illustrating a method for conducting a nucleicacid assay in accordance with embodiments of the present invention.

FIGS. 2A-2D are schematic illustrations of a DNA assay in accordancewith embodiments of the present invention.

FIG. 3A-3B is a schematic illustration of a detection region of afluid-holding device before (FIG. 3A) and after (FIG. 3B) sedimentationin accordance with an embodiment of the present invention.

FIG. 4 is a schematic illustration of a microfluidic disk arranged inaccordance with embodiments of the present invention.

FIGS. 5A-5C are schematic illustrations of an assay area 420 of amicrofluidic disk in accordance with an embodiment of the presentinvention.

FIGS. 6A-6B, 7A-7B, and 8A-8B are schematic illustrations of a detectionregion containing a sample fluid and a density media in accordance withan embodiment of the present invention.

FIG. 9 is a schematic illustration of a system according to anembodiment of the present invention.

FIG. 10 shows a dose response curve generated using serial dilutions ofDNA.

DETAILED DESCRIPTION

Certain details are set forth below to provide a sufficientunderstanding of embodiments of the invention. However, it will be clearto one skilled in the art that embodiments of the invention may bepracticed without various of these particular details. In someinstances, well-known chemical structures, chemical components,molecules, materials, electronic components, circuits, control signals,timing protocols, and software operations have not been shown in detailin order to avoid unnecessarily obscuring the described embodiments ofthe invention.

Embodiments of the present invention are directed toward systems,apparatus, and methods for detecting and/or quantifying cell-free DNA orother nucleic acids in a sample. As mentioned above, existing methodsfor detecting cell-free DNA in complex biological samples may becumbersome or suffer limited sensitivity. Examples according to thecurrent invention include analysis of target analytes including DNA,RNA, or other compositions of single or double stranded nucleic acids.References will be made herein to and examples given of applicationstargeting DNA, but it should be understood that in other examples, RNAor other compositions of single or double stranded nucleic acids may betargeted for detection and/or quantification.

Co-pending U.S. application Ser. No. 12/891,977, filed Sep. 28, 2010,entitled “Devices, systems, and methods for conducting sandwich assaysusing sedimentation,” is hereby incorporated by reference in itsentirety for any purpose. The aforementioned application includesexamples of the formation of complexes including a capture agent, targetanalyte, and labeling agent on sedimentation particles. Thus, targetagents may be separated from sample by affinity with a capture agent,and the sedimentation particles passed through a density medium.Examples of the present invention may capture nucleic acids directly toa surface of a sedimentation particle using the unique charge andchemical characteristics of nucleic acids. Accordingly, complexes formedon the sedimentation particle may simply include a target analyte (e.g.nucleic acid) and labeling agent. This separation mechanism mayadvantageously provide simple mechanisms for nucleic acid purificationand measurement.

FIG. 1 is a flowchart illustrating a method for conducting a nucleicacid assay in accordance with embodiments of the present invention. Inblock 110, complexes may be generated on sedimentation particles (e.g.beads) in a fluid sample. The complexes may include a target analyte(e.g. cell-free DNA) and labeling agent. Block 110 may be followed byblock 115. In block 115, the sedimentation particles may be transportedthrough a density media having a density lower than the beads but higherthan the fluid sample. Gravitational forces, generated naturally or bycentrifugation, may be used to transport the beads through the densitymedia. The sedimentation particles accordingly may sediment out of thefluid sample, forming a concentrated pellet. Block 115 may be followedby block 120. In block 120, signal may be detected from the labelingagent of the complexes. In some examples, by separating thesedimentation particles from the sample fluid using gravitationalforces, the beads are also concentrated, which may eliminate or reduce aneed for amplification of the labeling agent.

In block 110, complexes including the target analyte (e.g. cell-freeDNA), and labeling agent may be formed on beads in a fluid sample. Anysedimentation particles with appropriate surface properties, includingbeads, may be used, including but not limited to, polystyrene beads orsilica beads. Substantially any bead radii may be used. Examples ofbeads may include beads having a radius ranging from 150 nanometers to10 microns.

In nucleotide quantification sedimentation assays, the beads may havesurface modifications to enhance nucleotide capture. Similarly, thelabeling agent may be any suitable labeling agent for binding to thetarget nucleotide and providing a detection signal. Examples includechemical dyes with high binding affinity for DNA and/or other nucleicacids (e.g. nucleic acid dyes). Fluorescent labels including theaforementioned chemical dyes may provide an optical detection signal,however colorimetric or radioactive tags may also be used.

In examples of nucleic acid (e.g. cell-free DNA) assays, thesedimentation particles may be implemented as silica microparticles. Thesedimentation particles used in nucleic acid assays may advantageouslyhave a positively-charged surface. For example, the surfaces of thesedimentation particles (e.g. beads) may be amine-modified. In oneexample, 1 μm silica microparticles are used. In examples of cell-freeDNA assays, the complexes may be attached to the sedimentation particles(e.g. beads, silica microparticles) due to interaction between thepositively charged particle surface and negatively-charged nucleic acid.

In examples of nucleic acid assays, the sample may include (eitheroriginally or after mixing) a buffer in which nucleic acids may remainnegatively charged while proteins may generally maintain a net neutralor positive charge. For example, a high salt and low pH binding buffermay be used (e.g. 1M sodium sulfate and 1M sodium acetate, pH 3). Insome examples, the fluid sample may be configured to have an acidic pH(e.g. less than 4, less than 3.5, less than 3 in some examples). Theacidic fluid may advantageously cause proteins or other potentiallycontaminating components to have a positive charge while nucleic acidsmay maintain a negative charge. Generally, the buffer may include salts(e.g. kosmotropes, or order-forming salts) such that the nucleic acidsbind to the silica surfaces of the sedimentation particles (e.g. beads).By ordering water and thus increasing the hydrophobic effect,kosmotropes may facilitate protein aggregation to the exclusion of othermolecules such as nucleic acids, further decreasing the likelihood ofassay interference by proteins. In other examples, other binding buffersincluding, but not limited to chaotropic salts or PBS, may be used tobind the nucleic acids (e.g. DNA) to a negatively charged silicasurface.

In some examples, a negatively charged bead surface may be obtained, forexample, through use of untreated silica particles (e.g. microparticlesas formed by standard manufacturing practices prior tofunctionalization), silica particles treated with strong acids, throughfunctionalization of the silica surface with negatively charged moieties(e.g. carboxylic acids, phosphates), or through the use of polymericmicroparticles with negatively charged surface moieties (e.g. carboxylicacid functionalized polystyrene microparticles). In the case ofchaotropic salts (e.g. guanidine hydrochloride, potassium thiocyanate),the interaction may not be a direct binding to the surface of the beadsuch as the case of kosmotropic salts but rather an indirect binding.For example, and without being bound by theory, the positive ion of achaotropic salt may allow interaction between the surface of thenegatively charged bead and the negatively charged nucleic acid byforming a salt bridge (e.g. the positive ion may mediate the interactionwith the surface of the bead and the nucleic acid thus preventingrepulsion due to like-charge interactions). Additionally, chaotropicsalts may disrupt the order of water molecules surrounding nucleicacids, reducing hydrogen bonding and permitting a greater number of saltbridge formations. Other combinations or silica surface charge andbinding buffer are possible and known to the art. When chaotropic saltsor PBS are used, the background may be higher and mean fluorescenceintensity of the signal may be lower, however.

In examples of nucleic acid (e.g. cell-free DNA) assays, the labelingagent may be implemented as a dye for staining the nucleic acids. Dyesthat bind to DNA or other target nucleic acids (e.g. nucleic acid dyes)with high affinity (e.g. binding constant <20 nM) are highly preferred.Conventional DNA dyes that bind more weakly to DNA may be removed frombeads during sedimentation, weakening observed signal. Suitable dyesinclude, but are not limited to, ethidium homodimer 1, ethidiumhomodimer 2, and SYBR® gold. The aforementioned dyes may exhibit largeincreases in quantum yield upon binding to the nucleic acids (e.g. DNA),which may improve detection.

FIGS. 2A-2D are schematic illustrations of a DNA assay in accordancewith embodiments of the present invention. Although a DNA assay isshown, as mentioned above, any nucleic acids may be detected and/orquantified in other examples. In FIG. 2A, a sample 202 is shownincluding DNA, e.g. DNA 205. Samples used in DNA assays may or may notinclude DNA, as the assay may be used to detect the presence of DNAand/or to quantify the amount of DNA present in a sample. As describedabove, the sample may include any of a variety of fluids, includingbiological fluids such as serum.

FIG. 2B is a schematic illustration of a suspension of sedimentationparticles in accordance to an embodiment of the present invention. InFIG. 2B, a particle suspension 210 may include sedimentation particles(e.g. bead 212) and labeling agents (e.g. dye 214). As has beendescribed above, the sedimentation particles may have a positive charge(e.g. amine-treated silica particles). In other examples, thesedimentation particles may have a negative charge. The dye may beconfigured to stain DNA or other nucleic acids. As shown in FIG. 2A-2D,the sample and the particle suspension may be mixed (e.g. by vortexing,pipeting, and/or sonication). Either the sample, the particlesuspension, or both, may contain salts as described above such that theDNA, if present in the mixture, may have a negative charge whileproteins that may be present in the sample may have a positive orneutral charge. In other examples, salts may be present to facilitatenegatively-charged nucleic acids forming complexes with beads having anegatively-charged surface. Such binding may make use of salt bridges,as discussed above.

FIG. 2C is a schematic illustration of complexes that may be formed inthe mixture. DNA 205 may be labeled with the dye 214 and form a complexwith the positively-charged bead 212. Any number of complexes may beformed on the bead, with four shown in FIG. 2C. The mixture 220 may belayered over a density medium 222. The density media 222 is generally aliquid which may have a density lower than a density of sedimentationparticles (e.g. beads) used in an assay and higher than a density of thefluid sample. The density media may generally be implemented using afluid having a density selected to be in the appropriate range—lowerthan a density of the beads used to conduct an assay and higher than adensity of the fluid sample. In some examples, a fluid sample may bediluted for use with a particular density media. The density media mayinclude, for example, a salt solution containing a suspension of silicaparticles which may be coated with a biocompatible coating. An exampleof a suitable density media is Percoll™, available from GE Lifesciences.Particular densities may be achieved by adjusting a percentage ofPercoll™ in the salt solution. More generally, viscosity and density maybe adjusted by changing a composition of the media. Varying theconcentration of solutes such as, but not limited to, sucrose ordextran, in the density media, may adjust the density and/or viscosityof the media. The mixture 220 may be introduced into a fluidic feature(e.g. microfluidic device, including those described herein, vial, orother fluid-containing structure) such that the mixture 220 is next toor on top of the density media 222. Before or after the layering, themixture may be incubated to allow for formation of the complexes. Thedensity media may have a density that is greater than the sample butless dense than the sedimentation particles (e.g. bead 212).Sedimentation forces (e.g. centrifugal or gravitational) may be appliedto the mixture. For example, the feature containing the mixture may bespun to apply a centrifugal force.

FIG. 2D is a schematic illustration of the fluid containing structurefollowing sedimentation. The sedimentation particles, including the bead212 may be transported through the density media 222 responsive to thesedimentation forces and may form a pellet 226 at an end of thestructure. As the sedimentation particles (e.g. beads) are transportedthrough the density media, the flow may wash the particles, removingunbound material and improving the detection of the assay. Moreover, thedensity media may be buffered with PBS which may enhance a fluorescentsignal of DNA-bound dye. Sample and any unbound label may not betransported through the density media 222 and may remain within thesample or at an interface between the density media and the sample.Moreover, any red blood cells present in a whole blood sample may beless dense than the density media, and the cells may also remain at theinterface between the density media and the sample.

Nucleic acid assays conducted in accordance with embodiments of thepresent invention may accordingly separate interfering matrix componentsfrom the nucleic acids in complexes. For example, proteins or othercomponents with a positive or neutral charge may not form complexes witha positively-charged sedimentation particle. Other unbound particles(e.g. whole blood cells) may not travel through the density media due tothe density of the particle being less than the media. Accordingly,separation may be improved. The sensitivity of detection may also beimproved relative to standard techniques by the accurate separation,concentration in a pellet, and/or enhancement of the dye signal duringtransport through a PBS buffered density medium. Still further DNAassays in accordance with the present invention may be performed in arelatively short period of time, less than 10 minutes in some examples.

It may be of interest to directly measure cell free DNA or nucleic acidlevels in whole blood samples. FIG. 3A-3B is a schematic illustration ofa detection region 330 of a fluid-holding device (e.g. microfluidicdevice, disk) before (FIG. 3A) and after (FIG. 3B) sedimentation inaccordance with an embodiment of the present invention. A whole bloodsample 302 including red blood cells 305 and silica beads 315 may beintroduced to the detection region 330 next to or over a density media310. Although not shown in FIG. 3A-3B, the silica beads 315 may bemodified with positively charged surface molecules such as amines toeffectively bind nucleic acids in the fluid sample 302, as generallydescribed above. The density media 310 may have a density greater thanthe red blood cells 305, but less than that of the silica beads 315.

Generally, blood cells may have a density less than or equal to 1.095g/cm³, and the silica beads may have a density of about 2.05 g/cm³.Accordingly, the density media 310 may have a density of between about1.095 g/cm³ and 2.05 g/cm³. In one example, the density media 310 has adensity of 1.11 g/cm³.

Sedimentation may occur under the influence of a natural gravitationalfield, such as by allowing the assay to sit, unpowered, under theinfluence of a gravitational field. Sedimentation may also occur usingcentrifugal force, such as by spinning a microfluidic disk. The use of agravitational field may be preferred over a centrifugal force inembodiments where powered centrifugal force may be undesirable, such asin a circulating DNA test for first responders. For example, silicabeads on the order of 10-30 microns in diameter may sediment in minutesunder a normal gravitational field. Following sedimentation, as shown inFIG. 3B, the blood cells 305 may be prohibited from transport throughthe denser density media 310. The silica beads 315, however, may betransported through the density media 310 to a detection location, andDNA may be detected using signal from a labeling agent, as describedabove.

In some embodiments, methods described herein may be conducted within amicrofluidic disk. FIG. 4 is a schematic illustration of a microfluidicdisk 400 arranged in accordance with embodiments of the presentinvention. The microfluidic disk 400 may include a substrate 410 whichmay at least partially define regions of assay areas 420, 421, 422, and423. The microfluidic disk 400 may include a fluid inlet port 425 influid communication with the assay areas 420, 421, 422, and 423. Eachassay area may include any of a variety of fluidic features andcomponents, including but not limited to, channels, chambers, valves,pumps, etc. As shown in FIG. 4, each assay area includes a mixingchamber and a detection region connected by a channel containing avalve. During operation, as will be described further below, fluidsincluding sample liquids, density media, and/or beads suspended in afluid, may be transported using centrifugal force from an interior ofthe microfluidic disk 400 toward a periphery of the microfluidic disk400 in a direction indicated by an arrow 430. The centrifugal force maybe generated by rotating the microfluidic disk 400 in the directionindicated by the arrow 435, or in the opposite direction.

The substrate 410 may be implemented using any of a variety of suitablesubstrate materials. In some embodiments, the substrate may be a solidtransparent material. Transparent plastics, quartz, glass, fused-silica,PDMS, and other transparent substrates may be desired in someembodiments to allow optical observation of sample within the channelsand chambers of the disk 400. In some embodiments, however, opaqueplastic, metal or semiconductor substrates may be used. In someembodiments, multiple materials may be used to implement the substrate410. The substrate 410 may include surface treatments or other coatings,which may in some embodiments enhance compatibility with fluids placedon the substrate 410. In some embodiments surface treatments or othercoatings may be provided to control fluid interaction with the substrate410. While shown as a round disk in FIG. 4, the substrate 410 may takesubstantially any shape, including square.

In some embodiments, as will be described further below, the substrate410 may itself be coupled to a motor for rotation. In some embodiments,the substrate may be mounted on another substrate or base for rotation.For example, a microfluidic chip fabricated at least partially in asubstrate may be mounted on another substrate for spinning. In someexamples, the microfluidic chip may be disposable while the substrate orbase it is mounted on may be reusable. In some examples, the entire discmay be disposable. In some examples, a disposable cartridge includingone or more microfluidic channels may be inserted into disk or othermechanical rotor that forms part of a detection system.

The substrate 410 may generally, at least partially, define a variety offluidic features. The fluidic features may be microfluidic features.Generally, microfluidic, as used herein, refers to a system, device, orfeature having a dimension of around 1 mm or less and suitable for atleast partially containing a fluid. In some embodiments, 500 μm or less.In some embodiments, the microfluidic features may have a dimension ofaround 100 μm or less. Other dimensions may be used. The substrate 410may define one or more fluidic features, including any number ofchannels, chambers, inlet/outlet ports, or other features.

Microscale fabrication techniques, generally known in the art, may beutilized to fabricate the microfluidic disk 400. The microscalefabrication techniques employed to fabricate the disk 400 may include,for example, embossing, etching, injection molding, surface treatments,photolithography, bonding and other techniques.

A fluid inlet port 425 may be provided to receive a fluid that may beanalyzed using the microfluidic disk 400. The fluid inlet port 425 mayhave generally any configuration, and a fluid sample may enter the fluidinlet port 425 utilizing substantially any fluid transport mechanism,including pipetting, pumping, or capillary action. The fluid inlet port425 may take substantially any shape. Generally, the fluid inlet port425 is in fluid communication with at least one assay area 420, and maybe in fluid communication with multiple assay areas 420-423 in FIG. 4.Generally, by fluid communication it is meant that a fluid may flow fromone area to the other, either freely or using one or more transportforces and/or valves, and with or without flowing through interveningstructures.

The assay area 420 will be described further below, and generally mayinclude one or more channels in fluid communication with the fluid inletport 425. Although four assay areas 420-423 are shown in FIG. 4,generally any number may be present on the microfluidic disk 400.

As the microfluidic disk 400 is rotated in the direction indicated bythe arrow 435 (or in the opposite direction), a centrifugal force may begenerated. The centrifugal force may generally transport fluid from theinlet port 425 into one or more of the assay areas 420-423.

Accordingly, the microfluidic disk 400 may be used to perform assaysdescribed herein. Centrifugal forces may be used to generatesedimentation forces described herein. In other examples, however,gravity may be used to generate sedimentation forces, and assaysdescribed herein may be conducted in a vial or other container.

FIGS. 5A-5C are schematic illustrations of an assay area 420 of amicrofluidic disk in accordance with an embodiment of the presentinvention. The assay area 420 includes a channel 510 in fluidcommunication with the fluid inlet port 425. The channel 510 may be influid communication with a detection region 530, which may beimplemented using the detection region 330 of FIG. 3A-3B. Anotherreservoir 535 may be in fluid communication with the detection region530 via a channel 540, which may include or serve as a valve. Thedetection region 530 may be implemented as a fluidic chamber, channel,or reservoir, and detection may occur at an end of the region. In otherexamples, detection may not occur in the detection region 530, but thesample may be transported elsewhere for detection.

The detection region 530 and reservoir 535 may generally be implementedusing any size and shape, and may contain one or more reagents includingsolids and/or fluids which may interact with fluid entering and/orexiting the features.

The detection region 530 may be configured to contain a density media.Constituents of an appropriate density media are explained previously.In some embodiments, the density media may include a detergent, such asTween® 20. The detergent may enhance a wash function of transportthrough the density media, as will be described further below.

Sample, sedimentation particles, and dye may first be mixed and/orincubated in the reservoir 535, as shown in FIG. 5A, then introduced ata selected time to the detection region 530 by operation of the valve540. That is, a mixture of sample, sedimentation particles, and dyesuspension may be present in the reservoir 535 (for example byintroducing the different components separately, or by loading a mixturecontaining all components into the reservoir 535). The valve 540 may beclosed to contain the components in the reservoir 535 and allow thecomponents to incubate to form complexes.

On opening the valve 540, as shown in FIG. 5B, the mixture including thesample, beads, and dye, which may have formed complexes, may beintroduced to the detection region 530. In some examples, the reservoir535 may not be provided, and the mixture may be loaded directly into thedetection region 530, which may be a channel or chamber. Detection maytake place at an end of the detection region 530. The detection region530 may further have a tapered or other shape at the end of thedetection region 530, as was shown in FIG. 3A-3B.

The detection region 530 may be a channel or chamber and may vary inconfiguration in accordance with the detection technique employed, aswill be described further below. The detection region 530 may generallybe configured to allow for detection of a signal emitted by labelingagents in a complex. The complex may include cell-free DNA and labelingagent in embodiments pertaining to cell-free DNA assays. The complexesinclude sedimentation particles (e.g. beads).

Centrifugal forces may generally be used to transport the mixture fromthe inlet port 425 toward the detection region 530. Additionally,centrifugal forces may be used to transport density media from thereservoir 535 to the detection region 530. In other examples,pressure-driven or other flow drivers may be used to load fluids intothe device and transport the mixture to the detection region 530.

Incubation of sedimentation particles and labeling agents with thesample may take place within a microfluidic disk. Referring again toFIG. 5A, a fluid sample containing a target analyte may be introduced tothe inlet port 425 and provided to the reservoir 535 through the channel510. Any of a variety of suitable fluid samples may be used including,but not limited to, whole blood, buffer solutions, serum, or otherbiological fluid samples. Generally, the fluid sample will includetarget analytes to be detected in accordance with embodiments of thepresent invention. The fluid sample may contain beads designed to bindto nucleic acids and/or label agents designed to bind nucleic acids. Inother examples, the beads and/or label agents may be introduced to thefluid sample within the microfluidic disk 400. For example, a fluidcontaining the beads and/or label agents may be provided to a differentinlet port in fluid communication with the channel 510 of FIG. 5A-5C.Either by mixing components or by providing a fluid containing thecomponents, a sample fluid including beads, target analytes, andlabeling agents, may be transported to the detection region 530 of FIG.5A-5C. The transport of the sample fluid may occur through any type oftransport mechanism, including centrifugal force, pressure-driven flow,pumping, or other mechanisms. In other examples, beads having captureagents on their surface may be incubated with target analyte and/orlabeling agents prior to introduction to a microfluidic disk. In such anexample, complexes may be formed on beads in a sample fluid prior toproviding the sample fluid to the microfluidic disk.

The detection region 530 of FIG. 5A-5C may contain density media, ordensity media may be transported into the detection region 530 fromanother location, such as from the reservoir 535. The channel 540 mayhave a width selected to serve as a valve, such that a spin rate over athreshold amount is required to initiate a flow of the density mediafrom the reservoir 535 through the channel 540. In some examples, thechannel 540 has a width selected such that any spin rates used totransport sample into the reservoir 535 is insufficient to transport thesample into the detection region 530. A spin rate of the microfluidicdisk 400 may then be increased to initiate or enhance a flow of thesample from the reservoir 535 into the detection region 530. In thismanner, the channel 540 may function as a valve. Other valve structuressuch as wax plugs that melt at a known temperature may be used in otherexamples.

Once the mixture of sample, bead, and dye is in the detection region 530above the density media, sedimentation forces may be used to transportthe beads through the density media to form a bead pellet, as shown inFIG. 5C. If dye and target analytes are present on the bead, they may bedetected in the concentrated pellet.

Accordingly, a sample fluid including: 1) beads; 2) target analytes; and3) labeling agents may be transported to an interface with a densitymedia and sedimentation forces used to transport the beads, along withany bound complexes, through the density media to form a pellet at theend of the detection region.

FIGS. 6A-6B, 7A-7B, and 8A-8B are schematic illustrations of thedetection region 530 containing a sample fluid 805 and a density media810 in accordance with an embodiment of the present invention.Components of the sample fluid 805 are shown for purposes ofillustration beneath the detection region 530 in FIG. 8B. Schematicviews of the sample components, and complex formation (FIGS. 6B, 7B, and8B) are shown below the detection region view (FIGS. 6A, 7A, and 8A) forease of illustration. The sample fluid includes plurality of beads,including a bead 820 with positive surface charge. The sample fluid 805further includes target analytes, such as DNA 824, and labeling agents826 (FIG. 6B).

The sample fluid may then be incubated. FIG. 7A is a schematicillustration of the detection region 530 containing the sample fluid 805and the density media 810 following an incubation period. Complexes haveformed on the bead 820. The target analyte 824 has bound to the bead 820and labeling agent 826 (FIG. 7B). Some unbound, free labeling agents,however, remain in the sample fluid 805. A time of little to nocentrifugal force may be provided to allow for incubation. Additionally,in some examples, a region of the microfluidic disk containing thesample fluid may be heated to enhance incubation. In this manner,complexes may be formed on the beads. As understood in the art, theamount of labeling agent bound to complexes on the beads will generallybe proportional to the amount of target analyte in the fluid sample. Anynumber of complexes may be formed on the beads, with three complexesshown on the bead 820 in FIG. 7B.

The beads may then be transported through the density media. The beadsare transported through the density media using centrifugal force, suchas that which may be applied by a motor, described further below.Following a period of centrifugal force, the beads may be concentratedin a detection location. FIG. 8A is a schematic illustration of thedetection region 530 following transport of beads through the densitymedia. The sample fluid 805 may remain separated from the density media810, as the density media 810 may have a density higher than that of thesample fluid 805. Free, unbound labeling agent, may remain in the samplefluid 805. Beads 820, including complexes, may be transported throughthe density media 810 to a detection location 830. Beads at thedetection location 830 are shown for purposes of illustration under thedetection region 530 in FIG. 8B. The bead 820 includes complexescontaining target analyte 824 and labeling agent 826. However, unboundlabeling agent may not be found in the detection location 830. As shownin FIGS. 6A-6B, 7A-7B, and 8A-8B, centrifugal force may accordingly beused to separate complexed beads from a sample fluid and to concentratethe complexed beads. In this manner, the need for additional wash andamplification steps in a nucleic acid quantification assay may bereduced or eliminated. Signal from labeling agent of the concentratedbeads may be detected from the detection location 830 using, forexample, the detection module examples described below.

FIG. 9 is a schematic illustration of a system according to anembodiment of the present invention. The system 900 may include the disk400 of FIG. 4 with one or more assay areas 420. A motor 905 may becoupled to the disk 400 and configured to spin the disk 400, generatingcentrifugal forces. A detection module 910 may be positioned to detectsignal from labeling agents in a detection region of the assay area 420,as will be described further below. An actuator 915 may be coupled tothe detection module 910 and configured to move the detection modulealong the detection region in some examples. A processing device 920 maybe coupled to the motor 905, the detection module 910, and/or theactuator 915 and may provide control signals to those components. Theprocessing device 920 may further receive electronic signals from thedetection module 910 corresponding to the labeling agent signalsreceived by the detection module 910. All or selected components shownin FIG. 9 may be housed in a common housing in some examples.Microfluidic disks, which may be disposable, may be placed on the motor905 and removed, such that multiple disks may be analyzed by the system900.

The motor 905 may be implemented using a centrifugation and/or steppermotor. The motor 905 may be positioned relative to the detection module910 such that, when the disk 400 is situated on the motor 905, the diskis positioned such that a detection region of the assay area 420 isexposed to the detection module 910.

The detection module 910 may include a detector suitable for detectingsignal from labeling agents in complexes that may include labelingagent. In nucleic acid (e.g. cell-free DNA) assays, the complexes mayinclude DNA and labeling agent. The complexes may be formed on thesurface of one or more sedimentation particles (e.g. beads), asdescribed further below. The detector may include, for example, a laserand optics suitable for optical detection of fluorescence fromfluorescent labeling agents. The detection module may include one ormore photomultiplier tubes. In other examples, other detectors, such asphotodiodes or CCD cameras, may be used. The actuator 915 may move thedetector in some examples where signal may be detected from a variety oflocations of the microfluidic disk 400, as will be described furtherbelow.

The processing device 920 may include one or more processing units, suchas one or more processors. In some examples, the processing device 920may include a controller, logic circuitry, and/or software forperforming functionalities described herein. The processing device 920may be coupled to one or more memories, input devices, and/or outputdevices including, but not limited to, disk drives, keyboards, mice, anddisplays. The processing device may provide control signals to the motor905 to rotate the disk 400 at selected speeds for selected times, aswill be described further below. The processing device may providecontrol signals to the detection module 910, including one or moredetectors and/or actuators, to detect signals from the label moietiesand/or move the detector to particular locations, as will be describedfurther below. The processing device may develop these control signalsin accordance with input from an operator and/or in accordance withsoftware including instructions encoded in one or more memories, wherethe instructions, when executed by one or more processing units, maycause the processing device to output a predetermined sequence ofcontrol signals. The processing device 920 may receive electronicsignals from the detection module 910 indicative of the detected signalfrom labeling agents. The processing device 920 may detect a targetanalyte and/or calculate a quantity of a target analyte in a fluidsample based on the signals received from the detection module 910, aswill be described further below. Accordingly, the processing device 920may perform calculations as will be described further below. Thecalculations may be performed in accordance with software including oneor more executable instructions stored on a memory causing theprocessing device to perform the calculations. Results may be stored inmemory, communicated over a network, and/or displayed. It is to beunderstood that the configuration of the processing device 920 andrelated components is quite flexible, and any of a variety of computingsystems may be used including server systems, desktops, laptops,controllers, and the like.

Having described examples of microfluidic disks and systems inaccordance with embodiments of the present invention, methods forconducting assays will now be described. Some discussion will also beprovided regarding mechanisms for sedimentation and centrifugation. Thediscussion regarding mechanisms is provided as an aid to understandingexamples of the present invention, but is in no way intended to limitembodiments of the present invention. That is, embodiments of thepresent invention may not employ the described mechanisms.

Sedimentation of spheres may occur within a viscous fluid under theinfluence of a gravitational field (which may be natural or induced bycentrifugation). The settling velocity of approximately sphericalparticles may be described by Stoke's flow equations:

${V_{s} = {\frac{2}{9}\frac{\left( {\rho_{p} - \rho_{f}} \right)}{\mu}{gR}^{2}}};$

where V_(s) is the sedimentation velocity, μ is the fluid viscosity,ρ_(p) is the density of the particle, ρ_(f) is the density of the fluid,g is acceleration due to effective gravity, and R is the particleradius. Note that sedimentation rate scales with the square of particleradius and therefore a small difference in radius may form a basis forseparation of particles in some examples, as they may sediment at adifferent rate. There is also a linear dependence of sedimentation ratewith the difference in density between the particle and the surroundingfluid, which may also be an effective mechanism for separation.Accordingly, beads or other particles may be separated according totheir density and/or radius based on different sedimentation velocities.Separation of particles using these principles may be referred to as“rate zonal centrifugation.”

For nanometer scale particles, such as nucleic acids, gravitationalforces may act in conjunction with Brownian diffusion, but neither willgenerally cause motion of these nanometer scale particles oversignificant distances during typical centrifugal conditions (<100,000g). Accordingly, beads with affinity for nucleic acids may be used toseparate nucleic acids from a fluid sample containing mixture of othersmall molecules. By capturing nucleic acids on the bead surface, andseparating the beads from the remaining sample using sedimentation (e.g.centrifugal or gravitational) forces, the need for wash steps may bereduced or eliminated, because unbound labeling agents and/or othermolecules may be dissociated from the beads by fluid flow.

Example

1 mL of binding buffer (1M Na₂SO₄, 1M NaCH₃CO₂, pH 3) may be added to 50mg of 1 μm amine-modified silica microparticles. The substances may bemixed by vortexing, pipeting, and sonication to achieve a uniformsuspension of particles. 1 μL of 2000×SYBR® Gold may be added to resultin a particle suspension for use in a DNA assay. The mixture may beincubated with a sample for 10 minutes at room temperature protectedfrom light with mixing. 4 μL of the suspension may be added to a channelpreloaded with 3.5 μL of Percoll®. The channel may be spun at 8000 rpmfor 45 s, transporting the microparticles through the Percoll® to form apellet in a detection area. The pellet may be imaged using a fluorescentmicroscope with excitation 485 nm and emission 535 nm.

The sample may have 2 μL of serum. FIG. 10 shows a dose response curvegenerated using serial dilutions of DNA. A limit of detection wascalculated to be 1.1 ng/mL, and a limit of quantitation was calculatedto be 36.5 ng/mL. The normal range of cell-free DNA in health samplesmay be between 65-877 ng/mL, accordingly, these detection andquantitation limits are within a range useful for clinical significance.Capture efficiency was found to be greater than 99 percent in someexamples. 1000 ng was spiked into 1 mL of sample and captured usingamino-modified silica microparticles. After 10 minutes, SYBR® gold wasadded and the microparticles sedimented. Approximately 3.4 ng remainedunbound to the particles.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

What is claimed is:
 1. A method of conducting an assay, the methodcomprising: generating complexes which bind to a plurality of beads in afluid sample, individual ones of the complexes comprising anegatively-charged target analyte bound, using a charge, directly to apositively charged surface of the bead, and a labeling agent bound tothe target analyte, wherein the generating occurs in the presence of abinding buffer configured to provide an acidic pH to maintain thenegative charge of the target analyte; transporting the plurality ofbeads including the complexes through a density media, wherein thedensity media has a density less than a density of the beads and greaterthan a density of the fluid sample, and wherein the transporting occurs,at least in part, by a sedimentation force; and detecting a signal fromthe labeling agents of the complexes.
 2. The method of claim 1, whereinthe target analyte includes a nucleic acid.
 3. The method of claim 2,wherein the labeling agent comprises a nucleic acid dye.
 4. The methodof claim 3 wherein the nucleic acid dye has a binding constant <20 nM.5. The method of claim 3, wherein the beads have a positively-chargedsurface in the fluid sample and the nucleic acid has a negative charge.6. The method of claim 5, wherein the fluid sample includes kosmotropesor order-forming salts.
 7. The method of claim 5, wherein the fluidsample has an acidic pH<4 in order to cause contaminating proteins toexhibit positive charge.
 8. The method of claim 3, wherein the fluidsample includes chaotropic salts.
 9. The method of claim 1, wherein saidfluid sample comprises whole blood, wherein the density media has adensity greater than a density of red blood cells, and wherein themethod further comprises: separating the red blood cells from theplurality of beads at least in part by applying a sedimentation force tothe fluid sample such that the red blood cells remain at an interfacebetween the density media and the fluid sample.
 10. The method of claim1, wherein a surface of the beads comprises amines.
 11. The method ofclaim 1, wherein the density media comprises a detergent.
 12. The methodof claim 2, wherein the nucleic acid is a cell-free DNA.
 13. The methodof claim 1, wherein the sedimentation force comprises a centrifugalforce.
 14. The method of claim 1, wherein the transporting forms apellet.
 15. The method of claim 1, wherein the density media comprisessucrose, dextran, or a buffered density media.
 16. The method of claim1, wherein the binding buffer comprises a high salt concentration and alow pH.
 17. The method of claim 1, wherein the acidic pH is less than 4.18. A method of conducting an assay, the method comprising: generatingcomplexes which bind to a plurality of beads in a fluid sample,individual ones of the complexes comprising a negatively-charged targetanalyte bound, using a charge, directly to a positively charged surfaceof the bead, and a labeling agent bound to the target analyte, whereinthe generating occurs in the presence of a binding buffer configured toprovide an acidic pH to maintain the negative charge of the targetanalyte and a high salt concentration; transporting the plurality ofbeads including the complexes through a density media, wherein thedensity media has a density less than a density of the beads and greaterthan a density of the fluid sample, and wherein the transporting occurs,at least in part, by a sedimentation force; and detecting a signal fromthe labeling agents of the complexes.